This invention relates generally to solid support reagents used for the synthesis of functionalized polynucleotides, and more particularly, to the synthesis of polynucleotides having a nitrogen atom located at the 3'-end.
The continued rapid development of non-isotopic polynucleotide probes, DNA/RNA amplification methods, and bioactive antisense and triplex synthetic reagents, has greatly increased the demand for chemically modified polynucleotides. One popular approach to polynucleotide modification is to introduce a primary aliphatic amine to one end of the polynucleotide, thereby making it possible to readily functionalize the polynucleotide with substituents containing electrophilic moieties, e.g., isothiocyanates or activated esters. Common substituents include fluorophores, enzymes, biotin, intercalators, cross-linkers, nucleic acid cleaving reagents, modifiers of cellular uptake, and the like.
The most effective and convenient method for the introduction of an nitrogen atom to an end of a synthetic polynucleotide is to use an appropriately fuctionalized synthesis support followed by selective cleavage of the nitrogen-functionalized polynucleotide from that support. A number of methods currently exist for synthesizing 3'-nitrogen functionalized polynucleotides using modified supports, however, all of these methods produce a racemic mixture of products and/or require non-standard automated polynucleotide synthesis reagents and/or procedures, thereby complicating the purification and/or synthesis of these compounds.
For the foregoing reasons, there is a need for a solid support reagent capable of synthesizing 3'-nitrogen functionalized polynucleotides in non-racemic preparations using standard polynucleotide synthesis reagents, systems and procedures. More specifically, a modified solid support which is stable to (i) polynucleotide synthesis capping reagents such as acetic anhydride and pyridine, (ii) oxidants such as iodine, (iii) medium-strength acids such as trichloroacetic acid, and (iv) phosphorylating agents such as phosphoramidites; while at the same time being labile to typical polynucleotide synthesis cleavage reagents such as ammonium hydroxide.